Clathrin-coated vesicles (CCVs) play a role in controlling a cell's interaction with its environment. They are responsible for receptor- mediated endocytosis (RME) and participate in the biogenesis of organelles of the endocytic and regulated secretory pathway. Thus CCV function is critical for cellular metabolism of nutrients and pathogens, for control of growth factor receptor expression and for hormonal secretion. Understanding the molecular basis for cellular control of CCV formation therefore has implications in treating such diverse illnesses as heart disease, infection, cancer and diabetes. Furthermore, CCVs are the prototype coated vesicle involved in intracellular transport. Hence, understanding regulation of their formation provides insight into general mechanisms of molecular traffic between cellular membranes and organelles. CCVs are formed by polymerization of the protein clathrin into a polyhedral lattice on the cytoplasmic surface of cellular membranes. This process depends on interaction with adaptor molecules, which localize clathrin assembly to cellular membranes and recognize the receptor cargo that is sequestered in CCVs when clathrin polymerizes. Work during the past funding periods of this project has focused on the properties of the clathrin heavy and light chain subunits that control self-assembly. This analysis will now be applied to understanding how clathrin assembly is regulated in the cell. Aim 1 will establish how adaptor interactions with clathrin are regulated in the cell by phosphorylation and will define the molecular mechanism by which adaptors stimulate clathrin assembly. During the past funding period we demonstrated that adaptor-clathrin interaction in the cell is controlled by phosphorylation. The regulatory kinase and phosphatase will now be identified and how adaptors trigger cellular clathrin assembly will be defined, taking advantage of our recent progress in understanding clathrin self-assembly. Aim 2 is to establish the mechanism and function or phosphorylation of the clathrin heavy chain subunit and to understand clathrin recruitment to membranes at the morphological level. Preliminary results suggest that clathrin heavy chain phosphorylation is related to ligand-induced RME and influences clathrin recruitment to membranes. Phosphorylation will be analyzed biochemically and recruitment defined morphologically. Aim 3 will determine the differential roles of clathrin light chains LCa and LCb as regulators of clathrin function in vivo. Genetic deletion will be used to define LCa and LCb function in mice, providing the first insight into tissue-specific and systemic roles of CCV-mediated membrane traffic.